Screening methods for compounds that modulate the activity of G-protein coupled receptors

ABSTRACT

The present invention relates to a screening system for modulators of GPCRs. Further it relates to recombinant vector systems for the heterologous expression of heterodimeric g-protein coupled receptors (GPCRs) in eucaryotic host cells. Preferably the functional expression of engineered GPCRs for the perception of sweet and L-amino acid taste or more preferably the use of said receptors for the identification of functional ligands is also encompassed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.11/810,402, filed Jun. 5, 2007. Co-pending U.S. application Ser. No.11/810,402 is hereby incorporated by reference herein in its entirety.This application further claims priority to its parent application,European Patent Application No. 06011710.8 filed Jun. 7, 2006 which isalso hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a screening method for theidentification of modulators (agonists as well as antagonists) ofselected GPCRs and the thus identified modulators. In a preferredembodiment of the present invention these modulators may be tastemodulators. The present invention relates further to recombinant vectorsystems for the stable, heterologous expression of selectedheterodimeric g-protein coupled receptors (GPCRs) in eucaryotic hostcells. The functional expression of engineered GPCRs for the perceptionof sweet and L-amino acid taste and the use of said receptors for theidentification of functional ligands is disclosed.

BACKGROUND OF THE INVENTION

In this specification, a number of documents are cited. The disclosureof these documents, including manufacturer's manuals and patentapplications or patents, is herewith incorporated by reference in itsentirety.

GPCRs represent the largest family of cell surface receptors with anestimated number of up to 1000 genes within the human genomecharacterized by a seven-transmembrane configuration as their mainfeature. (Bockaert and Pin, 1999; Pierce et al., 2002). GPCRs areactivated by a multitude of different ligands, including peptides,proteins, lipids, small molecules, ions or even photons. Activated GPCRsalter their conformation allowing it to catalyze the exchange ofguanosine diphosphate (GDP) for guanosine triphosphate (GTP) on theα-subunit of a heterotrimeric g-protein coupled to the GPCR.Heterotrimeric G-proteins composed of one out of 18 differentα-subunits, one out of 5 different β-subunits and one out of 11different γ-subunits are usually classified by the nature of theirα-subunit and generally grouped into four main classes: G_(s), whichactivates adenylyl cyclase; G_(i), which inhibits adenylyl cyclase;G_(q), which activates phospholipase C; and G_(12/13) with heterologousfunctions. In addition to the α-subunit dependent signaling theβ/γ-subunits can function as signaling molecules on their own. GPCRdependent signaling becomes even more complex if it is considered thatthese receptors can exist as homo-oligomeric or hetero-oligomericcomplexes. (George et al., 2002; Milligan et al., 2003; Salahpour etal., 2000). Hence, it is not surprising that GPCRs are responsible forthe regulation of a wide variety of different physiological processes.

Recently the role of GPCRs in human senses like vision, olfaction andtaste has been subject of intensified investigations. While theparticipation of the GPCR rhodopsin in visual sensing is one of the mostcomprehensively examined g-protein coupled receptor signaling examplesof the last 30 years (Maeda et al., 2003), the role of GPCRs inolfaction and bitter taste as well as sweet taste was discovered in the1990ies. (Buck and Axel, 1991; Firestein, 2001; Lindemann, 1996b;Lindemann, 2001).

The discovery of GPCR signaling in taste perception is closely connectedto the discovery of tastant specific signaling in vertebrate tastecells. In electrophysical and biochemical studies it was apparent thattastant derived signaling resulted in typical GPCR dependent secondmessenger induction, e.g. cyclic nucleosides (cAMP, cGMP), inositoltri-phosphate (IP3) or calcium. (Kinnamon und Cummings, 1992; Kinnamonund Margolskee, 1996; Lindemann, 1996a). The participation of GPCRs intaste perception was further approved by the finding of the g-proteingustducin specifically expressed in vertebrate taste cells. (McLaughlinet al., 1992; Wong et al., 1996). On the other hand it was known fromgenetic mouse studies that the ability to sense sweet taste of e.g.saccharin was linked to the so called sac locus on mouse chromosome 4.(Bachmanov et al., 2001; Lush, 1989; Lush et al., 1995). Based on thesedata it was obvious to search for GPCR sequence tags in taste cellderived subtracted cDNA libraries or by performing genomic sequencescanning to further narrow down the mouse sac locus for theidentification of GPCR analogs as putative taste receptors. These twoapproaches led to the rat, mouse and human receptor DNA sequences forthe taste GPCRs T1R1 and T1R2 (Hoon et al., 1999; Hoon and Ryba, 1997)as well as T1R3. (Kitagawa et al., 2001; Li et al., 2001; Max et al.,2001; Montmayeur et al., 2001; Sainz et al., 2001). Homology alignmentsrevealed that these taste receptors like the homodimeric metabotrophicglutamate receptor (mGluR), the heterodimeric γ-aminobutyric acid type Breceptor (GABA_(B)R) and homodimeric extracellular calcium receptors aremembers of the small family of class C GPCRs. As a common characteristicmost of the class C receptors exhibit a large extracellular aminoterminal domain composed of a so called venus flytrap module (VFTM) anda cysteine rich domain (CRD) that connects the VFTM to the heptahelicaldomain. (Pin et al., 2003). Besides that homo- as well ashetero-oligomerisation was described for several of these class Creceptors. (Bai et al., 1998; Kaupmann et al., 1998; Kunishima et al.,2000; White et al., 1998). Consequently, the characteristic feature ofGPCR oligomerisation of class C receptors was tested for the putativesweet taste receptors T1R1, T1R2 and T1R3.

By recombinant heterologous expression in eucaryotic cell systems afunctional expression and tastant specific activation of an artificiallylinked G-Protein dependent signaling cascade was demonstrated by calciumimaging. T1R receptors assemble to build up functional taste receptors.As a result of several investigations it was shown that theheterodimeric T1R1/T1R3 functions as a glutamate (umami) and L-aminoacid receptor whereas the heterodimeric T1R2/T1R3 functions as a highaffinity sugar and artificial sweetener receptor. Particularly,heterodimeric co-expression of T1R1 and T1R3 results in taste receptorsthat respond to umami taste and monosodium glutamate stimuli whereasheterodimeric co-expression of T1R2 and T1R3 results in taste receptorsthat respond to sweet stimuli like diverse sugars (e.g. glucose andsucrose), artificial sweetener (e.g. acesulfam K, cyclamat, saccharin)and sweet proteins like monellin, thaumatin, brazzein (Li et al., 2002;Nelson et al., 2002; Nelson et al., 2001; Zhao et al., 2002). A similarchronicle could be generated for the identification of GPCRs for theperception of bitter taste with the exception that so far no homo- oroligomerisation has been reported for these so called T2R-GPCRs.(Meyerhof et al., 2005).

The above discussed identification of genes coding for receptorsresponsible e.g. for taste perception, together with cloning said genesinto appropriate vectors for the expression of said proteins ineukaryotic cells and the transformation of said cells with said vectorsraised the expectation that screening systems and/or screening methodsfor GPCR modulators, i.e. agonists and antagonists of the above detailedreceptors should be easy to be developed within a reasonable time.

This is reflected by a huge and still growing number of patentapplications in this field.

The cloning of T1R1 is disclosed in different patent applications, e.g.in WO 03/025137; in WO 00/06952 (wherein it is designated GPCR-B3)US020040191862A1 and WO2005/033125.

The cloning of T1R2 is disclosed in patent applications WO 03/025137,US020040191862A1 and US020030040045A1

The cloning of T1R3 is disclosed in patent applications WO 03/025137, WO03/025137, US020040191862A1 and US020030040045A1

A system for the expression of said proteins in eukaryotic cells isdisclosed in patent applications WO 03/025137, WO 00/06952,US20040191862A1, WO2004069191 and US20030040045A1.

A screening system for putative taste modulators is disclosed in patentapplications WO 00/06952, WO2004069191 and US20030040045A1.

Yet, nothing is to be told about the successful identification of newmodulators, e.g. new artificial taste modulators such as new sweetenersutilizing such screening methods/systems.

The ongoing debate on obesity in developed countries and the growinghealth consciousness of consumers lead to an increasing demand of foodand beverages with significant calorie reduction compared to productsfully sweetened with carbohydrates such as sucrose, glucose, fructose orsyrups such as HFCS 55 or 42. As the consumer usually is not willing tocompromise on taste products should have similar sweetness intensity andtaste quality as products regularly sweetened with these carbohydrates.

High intensity sweeteners are substances, which have no or virtually nocalories and a sweetness potency several times higher than sugar. Highintensity sweeteners or blends of high intensity sweeteners are used infood and beverages to achieve a sweet taste without adding calories tothe products.

Most commonly used high intensity sweeteners are not from naturalorigin; They were discovered accidentally and are chemicallysynthesized. Most of them have a widespread approval in a large numberof countries. Examples are substances such as acesulfame K, alitame,aspartame, cyclamate, neohesperidine dihydrochalcone, neotame,saccharin, and sucralose.

However, no high-intensity sweetener matches the taste profile of sugarcompletely. They differ in characteristics such as sweetness profile,side taste and off-taste characteristics. Proper blending of differenthigh intensity sweeteners is known to overcome part of the tastelimitations of single high-intensity sweeteners. But even if a moresugar-like sweetness profile is achieved in products with high-intensitysweeteners only, they still can be distinguished sensorically from theircounterparts with just sugar or other carbohydrates by lack of mouthfeeland reduced flavour characteristics. Therefore a need exists for newhigh-intensity sweeteners which offer either alone or in blends withexisting sweeteners sweetness profiles and flavour characteristics muchcloser to sugar than the existing products can offer.

Besides calorie reduction many of today's consumers are seeking for foodand beverage products either without artificial additives or even beingfully organic. Theoretically natural high-intensity sweeteners couldfulfil this demand. A number of natural high-intensity sweeteners werediscovered throughout past years such as stevioside, rebaudioside,brazzein, thaumatin, mogroside, glycyrrhizin, monatin, abrusoside,monellin, phyllodulcin and others. These are substances which naturallyoccur in different plants and can be obtained by selective extractionmeasures. Besides very limited approvals and in some cases difficultiesto extract products on an industrial scale none of these products canclaim to offer a sugar-like taste. In fact, all of these substances showa sweetness with a far slower onset than sucrose and a very lingeringsweetness. Most of these products have strong side-taste and aftertastecharacteristics such as bitter, mentholic or liquorice notes or showeven strong cooling or numbing sensations. Therefore some of theseproducts, e.g. thaumatin, can be rather regarded as being flavourenhancer than sweetener. Blending of two or more of these substances cannot overcome these taste limitations. Therefore in the area of naturalsweetener the need for new high-intensity sweeteners with a tasteprofile closer to sugar is even stronger than in the case of artificialsweeteners (O'Brien Nabors, 2001; Leatherhead Food R A, 2000; Grenby,1996; von Rymon Lipinski und Schiweck, 1991).

Therefore, there still exists a need in the art to identify and isolatenew substances which may be used as modulators of taste perception, e.g.as sweeteners.

Notwithstanding the above, because of the high importance of these GPCRsin vivo, and the many different functions associated with saidreceptors, it has to be assumed that many of the modulators of GPCRsthat might be identified by the method of the present invention may beof practical value.

Therefore, the availability of a simple and reliable screening systemfor modulators of said receptors would be of big importance.

In multicistronic expression vectors the coding sequences of differentproteins are under the control of only one promoter and the differentcistrons are connected via virus derived internal ribosomal entry sites(IRES) or cap independent translation enhancer (CITE). IRES or CITEelements confer a translation initiation independent from the otherwisenecessary 5′-end of a messenger RNA, which is recognized by theeucaryotic ribosomes to start their scanning process for the firstaccessible translational start codon. (Fux et al., 2004; Hellen andSarnow, 2001). So far multicistronic expression vectors have beendescribed basically as dicistronic expression units for the coupledexpression of a gene of interest linked via an cap-independenttranslation initiation site to a resistance marker (conferringresistance to e.g. hygromycin, zeocin, neomycin) enabling selection ofstable cell lines for heterologous mammalian expression studies. Forthis approach IRES or CITE dependent expression vectors are commerciallyavailable.

Reports on genuine multicistronic expression studies in mammaliansystems with descriptions of tri-cistronic or even quadro-cistronicheterologous expression studies are rare and for the most part intendedto improve inducible protein expression e.g. for gene therapyapplications. However in this pioneering work multicistronic expressionshave been mostly performed with small and soluble proteins like reportergenes (green fluorescent protein, yellow fluorescent protein, redfluorescent proteins, secreted alkaline phosphatase, secreted amylase)or engineered transactivators e.g. for macrolide- or streptogramindependent expression or selection markers. Although these studies areaimed at potential therapeutic protein expression in gene therapyapplications, only few genes with a therapeutic potential are mentionedwithin this studies (e.g. vascular endothelial growth factor (VEGF); theoncoprotein bcl-2). (Fussenegger et al., 1998; Hartenbach andFussenegger, 2005; Kramer et al., 2003; Moser et al., 2000; Weber etal., 2005).

Concerning the expression of taste receptors there is one report ondicistronic expression of mouse taste receptors (mT2R8/5; mT1R3) eachfused to green fluorescent protein and linked via an IRES element to redfluorescent protein. This approach was applied to trace and localize theexpression pattern of taste receptors in neurons (Sugita and Shiba,2005).

SUMMARY OF ADVANTAGEOUS EMBODIMENTS OF THE INVENTION

Therefore, the technical problem underlying the present invention was toprovide a cell based screening system for modulators of GPCRs, i.e.establishing a method which makes it possible to successfully identifyimportant modulators of some selected GPCRs, preferably T1R-type tasteGPCRs. It was a further problem underlying the present invention toprovide means and methods for the isolation of identified modulatorswithout undue burden.

The solution to the above mentioned technical problems is achieved byproviding the embodiments characterized in the claims.

Therefore, specially preferred embodiments of the present invention aremeans and methods for the expression of heterodimeric T1R-type tasteGPCRs, wherein the expression of these heterodimeric T1R-type tasteGPCRs is effected by modulating the GPCR coding sequences as well astheir expression in multicistronic operons.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts different GPCR nucleotide sequences of the TIR class,presented in an alignment analysis, with the underlying distance matrixshown below;

FIG. 2 depicts an exemplary multicistronic eukaryotic expression vectorpTrix-Eb-R2R3;

FIG. 3 depicts a Ca^(z+)-assay with HEK_Ga15#17R2R3b #8 measuringseveral sweet tastants;

FIG. 4 depicts an alternative exemplary multicistronic eukaryoticexpression vector p Trix-Eb-R2R3.

DETAILED DESCRIPTION OF ADVANTAGEOUS EMBODIMENTS OF THE INVENTION

The above problem can surprisingly yet easily be solved by providing amethod for the identification of modulators of GPCRs, comprising thesteps of:

-   -   a. transforming eucaryotic host cells with genetic sequences        coding for one or more than one GPCR(s),    -   b. cultivation of the transformed host cells under conditions        sufficient to ensure the functional expression of said one or        more GPCRs,    -   c. contacting the cultivated host cell expressing the one or        more GPCRs in a functional manner with a potential modulator of        the selected one or more GPCRs,    -   d. measuring a specific cellular response on exposure of the        transformed host cell to the potential modulator, and    -   e. selecting an identificate, which induces a specific response.

Within the context of the present invention the term “modulator ofGPCRs” is intended to mean a substance, which if applied in vivo, would(a) trigger an intracellular response by binding to the selected GPCR or(b) would inhibit such a response in the presence of a sweet compound.E.g. in the case of sweet receptors as the selected GPCR this modulatorcould be a substance which tastes sweet or which potentiates thesweetness of a sweet tasting compound without being sweet on its own orwhich inhibits the sweetness of sweet substances.

The term “conditions sufficient to ensure the functional expression”shall be understood as a summary of all conditions needed to ensure thefunctional expression of the GPCRs, rather than cell culture conditions.In a preferred embodiment of the present invention this term pertains tothe vector used for transformation being a tricistronic vector. Further,this expression encompasses culturing cells which comprise a functionalg-.protein. Under any circumstances, the functional expression of theGPCR(s) must be ensured.

The terms “functional expression” or “expressing in a functional manner”means within the context of the present invention that the selectedreceptor maintains its ability to interact specifically with the samesubstances the receptor would interact with in the in vivo situation.

By “selected cellular response” e.g. changes in intracellular calciumlevels have to be understood, which can be measured easily e.g. by usingdyes like Calcium 3, Fura-2, Fluo-4, Indo-1 or the calcium dependentreporter protein aequorin. Any cellular response related to the activityof the selected GPCRs is encompassed. Further examples of measuring theactivity of GPCRs are (i) the activation of the second messenger cyclicadenosine monophosphate (cAMP) which can be quantified in GPCR dependentcell based assays with luminescent tags or with cAMP dependent reportergene expression (e.g. Luciferase, SEAP or similar) or with themelanophore technology using frog skin cells; (ii) the measurement ofβ-arrestin binding to the targeted GPCR after ligand induced activationof the GPCR via bioluminescence resonance energy transfer (BRET)measurements or GFP-tagged β-arrestin movement imaging; (iii) measuringthe agonist occupation of a GPCR and respectively the activation of theassociated G-Protein, which can be quantified by using the radioactive,non-hydrolyzable analogue of GTP, [35S]GTPγS; (IV) the use of responseelement controlled reporter gene assays for the readout of GPCRactivated interconnected pathways, including those involving MAPkinases, nonreceptor tyrosine kinases, receptor tyrosine kinases,phosphatidylinositol 3-kinases, and JNKs. (Eglen, 2005; Filmore, 2004;Milligan, 2003).

The term “identificate” is directed to a potential modulator after beingidentified as a true modulator of GPCR or the GPCR specific signalingcascade.

In a preferred embodiment the method is characterized in that the hostcell is selected from the group consisting of: HEK293 (human embryokidney), Hela (Human Negroid cervix epitheloid carcinoma), HT29 (HumanCaucasian colon adenocarcinoma grade II), A431 (human squamouscarcinoma), IMR 32 (human caucasian neuroblastoma), K562 (HumanCaucasian chronic myelogenous leukaemia), U937 (Human Caucasianhistiocytic lymphoma), MDA-MB-231 (Human Caucasian breastadenocarcinoma), SK-N-BE(2) (Human Caucasian neuroblastoma), SH-SY5Y(Human neuroblastoma), HL60 (human promyelocytic leukemia) or eukaryoticnon-human cell lines like CHO-K1 (Hamster Chinese ovary), COS-7 (MonkeyAfrican green kidney, SV40 transformed), S49 (mouse lymphoma), Ltk(Mouse C34/an connective tissue), NG108-15 (Mouse neuroblastoma×Ratglioma hybrid), B50 (Rat nervous tissue neuronal, ECACC), C6 (Rat glialtumour), Jurkat (Human leukaemic T cell lymphoblast), BHK (HamsterSyrian kidney), Neuro-2a (Mouse Albino neuroblastoma), NIH/3T3 (mouseembryo fibroblast), preferably HEK293 (human embryo kidney), Hela (HumanNegroid cervix epitheloid carcinoma), CHO-K1 (Hamster Chinese ovary) orNeuro-2a (Mouse Albino neuroblastoma).

It is essential that the host cell expresses a functional g-protein,preferably G-alpha15, either naturally or by means of genetic alterationof the host cell. Means for genetic alteration of eucaryotic cells arewell known in the art, and need not to be discussed in depth here. TheDNA-Sequences of G-proteins, e.g. G-alpha15 have already been describedin the art.

A further preferred embodiment of the method of the present invention ischaracterized in that the one or more GPCR(s) is (are) selected from thegroup consisting of T1R or T2R taste receptors, preferably a T1R-typeGPCR, especially T1R1, T1R2 and/or T1R3.

A further preferred embodiment of the method of the present invention ischaracterized in that two or more GPCRs are expressed in a heterologousco-expression of at least two different T1R-type GPCRs, preferablyT1R1/T1R3, more preferably T1R2/T1R3.

A further preferred embodiment of the method of the present invention ischaracterized in that the transformation is accomplished with amulticistronic vector, preferably a tricistronic vector.

The vector of the present invention is an expression vector. By the term“expression vector” it is meant that the vector is used to transform aselected eucaryotic host cell, which, after transformation, expressesthe gene or genes encoded by said vector. Expression vectors may be e.g.cloning vectors, binary vectors or integrating vector. Expressioncomprises transcription of the encoded nucleic acids into a functional(translatable) mRNA. Therefore, the respective control elements shouldbe present, e.g. a sequence promoting transcription of the messenger (apromoter) and (optionally) a polyadenylation signal. Means for theexpression of heterologous genes in eukaryotic cells are very wellstudied.

For the propagation of such vectors, usually prokaryotic cells such asE. coli are used. Therefore, although the vectors of the presentinvention are designed to work as expression vectors in eukaryoticcells, they also carry elements for propagation in prokaryotic cells,e.g. an origin of replication (ori) and an antibiotics resistance gene,e.g. amp^(r), kan^(r) and similar. Means for the propagation of vectorsin prokaryotes are well known in the art.

A list of possible eukaryotic expression vectors usable in the presentinvention, optionally usable for propagation in a prokaryotic host, andalready commercially available comprises: pCR1000, pCDM8, pcDNA1,pcDNA1.1, pcDNA1/Amp, pcDNA1.1/Amp and pcDNA1/Neo, pcDNA3, pcDNA3.1,pcDNA3.2, pcDNA6.2, pDEST26, pDEST27, pCR3.1, pcDNA3.1 His, pDisplay(Invitrogen); pTriEx (pTriEx-2-Hygro) (Novagen); pSI, pCI (pCI-neo),pTargeT (Promega); pERV3, pFB-ERV, pCFB-EGSH, pDual, pCMV-Script(Stratagene); pNEBR (New England Biolabs), pEAK (Edge Biosystems).

A further preferred embodiment of the method of the present invention ischaracterized in that the multicistronic vector comprises amulticistronic expression unit comprising downstream from a promoter forthe expression in an eucaryotic host and functionally linked thereto,the following cistrons:

-   -   a. GPCR₁    -   b. GPCR₂ and    -   c. a selection marker,        wherein the promoter preferably is a strong promoter suitable        for use in the selected host cell, more preferably being        selected from the group consisting of cytomegalovirus promoter        (P-CMV), human elongation factor 1 alpha promoter (P-E1α), human        ubiquitin promoter (P-ubi), simian virus promoter (P-SV40), Rous        sarcoma virus long terminal repeat promoter (P-RSV-LTR) and        similar, wherein the GPCR₁ and the GPCR₂ are independently from        another being selected from the group consisting of T1R or T2R        taste receptors, preferably of the group of T1R receptors, more        preferably a combination of T1R1-T1R3 or T1R2-T1R3, wherein the        selection marker is being selected from the group consisting of        hygromycin^(r), zeocin^(r), neomycin^(r), blasticidin^(r) or        puromycin^(r), and wherein both the GPCR1 and the GPCR2 as well        as the selection marker are functionally connected by        intervening IRES selected from the group consisting of        IRES_(EMCV), derived from encephalomyocarditis virus (synonym:        CITE_(EMCV)); IRES_(GTX), derived from the GTX homeodomain mRNA;        IRES_(Rbm3), derived from cold-inducible Rbm3; IRES_(PV),        derived of polioviral origin, IRES_(RV), derived from        rhinovirus, IRESFMDV, derived from food and mouth disease virus;        IRE_(HV), derived from hepatitis C virus, IRES_(CSFV), derived        from classic swine fever virus, IRES_(BVDV), derived from bovine        viral diarrhea virus; IRES_(FMLV), derived from friend murine        leukemia virus gag mRNA; IRES_(MMLV), derived from moloney        murine leukemia virus gag mRNA; IRES_(HIV), derived from human        immunodefiency virus env mRNA; IRES_(PSIV), derived from Plautia        stali intestine virus; IRES_(RPV), derived from Rhopalosiphum        padi virus; IRES_(KSH), derived from Karposi's        sarcoma-associated herpesvirus, preferably the IRES_(EMCV) being        derived from encephalomyocarditis virus (synonym: CITE_(EMCV))        and wherein the multicistronic expression unit is terminated by        a polyadenylation signal.

The term “functionally linked thereto” means within the context of thepresent invention that the components described are linked together tofunction in their intended manner.

A further preferred embodiment of the method of the present invention ischaracterized in that the multicistronic vector additionally comprises agenetic sequence coding for a g-protein or an equivalent thereof,preferably G-alpha 15 or an equivalent thereof, which is coded as anmonocistronic unit consisting of a promoter, the gene of the selectedg-protein and a polyadenylation site.

A further preferred embodiment of the method of the present invention ischaracterized in that the multicistronic vector additionally comprises agenetic sequence coding for a g-protein or an equivalent thereof,preferably G-alpha 15 or an equivalent thereof, which is coded as afourth cistron in a quadrocistronic arrangement via an additional IRESelement.

A further preferred embodiment of the method of the present invention ischaracterized in that the multicistronic vector additionally comprises agenetic sequence coding for a g-protein or an equivalent thereof,preferably G-alpha 15 or an equivalent thereof, the g-protein preferablybeing fused in-frame to above GPCR₁ and/or GPCR₂.

According to the present invention G proteins such as G-alpha15 orG-alpha16 or other promiscuous G proteins or G protein variants, or anendogenous G protein like gustducin, or another g-protein that whenexpressed in association with the multicistronically encoded GPCR(s)produces a functional read out may be used. In addition, G-beta andG-gamma proteins may also be used.

Subvariants of G-alpha 15 and/or G-alpha 16 with modified N-termini arealso well known in the art, and can be used accordingly.

Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as G-protein coupled receptor modulators can be anysmall chemical compound, or biological entity (e.g., protein, sugar,nucleic acid, lipid). Test compounds will typically be small chemicalmolecules and peptides. Generally, the compounds used as potentialmodulators can be dissolved in aqueous or organic (e.g., DMSO-based)solutions. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource. Assays are typically run in parallel, for example, in microtiterformats on microtiter plates in robotic assays. There are many suppliersof chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluke Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

So-called high throughput screening methods typically involve providinga combinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (e.g., ligand or modulator compounds).Such combinatorial chemical libraries or ligand libraries are thenscreened in one or more assays to identify those library members (e.g.,particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds so identified can serve asconventional lead compounds, or can themselves be used as potential oractual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressingan ion channel is attached to a solid phase substrate. In such highthroughput assays, it is possible to screen up to several thousanddifferent modulators or ligands in a single day. In particular, eachwell of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

In another aspect, the present invention encompasses screening and smallmolecule (e.g., drug) detection assays which involve the detection oridentification of small molecules that can bind to a given protein,i.e., a taste receptor polypeptide or peptide. Particularly preferredare assays suitable for high throughput screening methodologies.

In such binding-based detection, identification, or screening assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

Compounds which are identified according to the methods provided herein,and which modulate or regulate the biological activity or physiology ofthe T1R polypeptides according to the present invention are a preferredembodiment of this invention. It is contemplated that such modulatorycompounds may be employed as outlined above.

Assays that may be utilized with one or more T1Rs according to theinvention include by way of example assays that utilize a geneticselection for living cells; assays that utilize whole cells or membranefragments or purified taste receptor proteins; assays that utilizesecond messengers such as cAMP and IP3, assays that detect thetranslocation of arrestin to the cell surface, assays that detect theloss of receptor expression on the cell surface (internalization) bytested ligands, direct ligand-binding assays, competitive-binding assayswith inhibitors, assays using in vitro translated protein, assays thatdetect conformational changes upon the binding of a ligand (e.g., asevidenced by proteolysis, fluorescence, or NMR), behavioral assays thatutilize transgenic non-human animals that express a taste GPCR or acombination thereof, such as flies, worms, or mice, assays that utilizecells infected with recombinant viruses that contain taste GPCR genes,preferably measuring the change of intracellular calcium levels relativeto intracellular calcium levels without contacting the cell to themodulator.

A further preferred embodiment of the method of the present invention ischaracterized in that the change is an increase.

A further preferred embodiment of the method of the present invention ischaracterized in that the change is an increase relative tointracellular calcium levels when the cell is contacted by a modulatorin the presence of a sweet compound thereby enhancing the calcium levelabove the level of the level generated by the sweet compound alone. Inthis case the modulator can either be sweet on its own or either be atasteless compound without having an bioactive potential to activateT1Rs or combinations thereof on its own. The sweet compound preferablybeing selected from the group consisting of glucose, fructose,saccharose, acesulfam K, saccharin, cyclamat, aspartam, xylitol,stevioside, sucralose, thaumatin, monellin, brazzein, perillartine,glycyrrhizin, sucronic acid, P-4000, SC45647, NC174, neohesperidin andsweet tasting amino acids, more preferably selected from the groupconsisting of glucose, fructose, saccharose or xylitol.

A further preferred embodiment of the method of the present invention ischaracterized in that the change is a decrease relative to intracellularcalcium levels when the cell is contacted by a sweet compound instead ofthe modulator, the sweet compound preferably being selected from thenon-exclusive group consisting of glucose, fructose, saccharose,acesulfam K, saccharin, cyclamat, aspartam, xylitol, stevioside,sucralose, thaumatin, monellin, brazzein, perillartine, glycyrrhizin,sucronic acid, P-4000, SC45647, NC174, neohesperidin and sweet tastingamino acids.

A still further preferred embodiment of the present invention aremultiparameter optimized nucleic acid molecules coding for a GPCR,preferably a T1R-type GPCR, more preferably a T1R1, T1R2 or T1R3 or afunctionally equivalent receptor protein, and even more preferablycoding for proteins consisting of the amino acids sequences according toSEQ ID NO: 1 and/or SEQ ID NO: 2 or, optionally, functionally equivalentproteins.

These nucleic acid molecules in a preferred embodiment have the sequenceas depicted in SEQ ID NOs 3 and 4.

Nucleic acid molecules which are functionally equivalent to themolecules as depicted in SEQ ID NOs 3 and 4 are also encompassed by thepresent invention.

Within the context of the present invention, functionally equivalentproteins have the same or a very similar function in-vivo. Preferably,functionally equivalent proteins share at least 60%, more preferably atleast 80%, especially at least 90%, advantageously at least 99% identityin their amino acid sequence.

A functionally equivalent nucleic acid codes for a functionallyequivalent protein and it is optimized for expression in a eucaryoticcell. According to a multiparameter optimization the codon usage wasadapted to the codon bias of Homo sapiens genes. In addition, regions ofvery high (>80%) or very low (<30%) GC content have been avoided wherepossible. During the optimization process following cis-acting sequencemotifs were avoided:

a) internal TATA-boxes, chi-sites and ribosomal entry sitesb) AT-rich or GC-rich sequence stretchesc) ARE, INS, CRS sequence elementsd) repeat sequences and RNA secondary structures, as well ase) (cryptic) splice donor and acceptor sites, branch points.

The optimization process, starting from the human wild type sequences(wt_hT1R) revealed several sequences; surprisingly the sequences whichoffered the best performance (sh_T1R) in generating functional stablecell lines on the basis of the multicistronic expression approach werenot those with the theoretically optimal sequence (opt_hT1R) nor thewild type sequences. The differences of the here examplified sequencesare illustrated as a phylogenetic alignment (generated by thebioinformatic software clustal X) with an accompanying relationaldistance matrix in FIG. 1.

A very preferred embodiment of the present invention are multicistronicexpression vectors comprising more than one cistron coding for a GPCR.

The said multicistronic expression vector preferably comprisesdownstream from a promoter for the expression in an eucaryotic host celland functionally linked thereto, the following cistrons:

-   -   a. GPCR₁    -   b. GPCR₂ and a    -   c. selection marker,        wherein the promoter preferably is a strong promoter, more        preferably being selected from the group consisting of        cytomegalovirus promoter (P-CMV), human elongation factor 1        alpha promoter (P-E1α), human ubiquitin promoter (P-ubi), simian        virus promoter (P-SV40), Rous sarcoma virus long terminal repeat        promoter (P-RSV-LTR) and similar wherein the GPCR₁ and the GPCR₂        are independently from another being selected from the group        consisting of T1R or T2R taste receptors, preferably of the        group of T1R receptors, more preferably a combination of        T1R1-T1R3 or T1R2-T1R3, and wherein the selection marker is        being selected from the group consisting of hygromycin^(r),        zeocin^(r), neomycin^(r), blasticidin^(r) or puromycin^(r), and        wherein both the GPCR1 and the GPCR2 as well as the selection        marker are functionally connected by intervening IRES selected        from the group consisting of IRES_(EMCV), derived from        encephalomyocarditis virus (synonym: CITE_(EMCV)); IRES_(GTX),        derived from the GTX homeodomain mRNA; IRES_(Rbm3), derived from        cold-inducible Rbm3; IRES_(PV), derived of polioviral origin,        IRES_(RV), derived from rhinovirus, IRESFMDV, derived from food        and mouth disease virus; IRE_(HV), derived from hepatitis C        virus, IRES_(CSFV), derived from classic swine fever virus,        IRES_(BVDV), derived from bovine viral diarrhea virus;        IRES_(FMLV), derived from friend murine leukemia virus gag mRNA;        IRES_(MMLV), derived from moloney murine leukemia virus gag        mRNA; IRES_(HIV), derived from human immunodefiency virus env        mRNA; IRES_(PSIV), derived from Plautia stali intestine virus;        IRES_(RPV), derived from Rhopalosiphum padi virus; IRES_(KSH),        derived from Karposi's sarcoma-associated herpesvirus,        preferably, the IRES_(EMCV), derived from encephalomyocarditis        virus (synonym: CITE_(EMCV)) and wherein the multicistronic        expression unit is terminated by a polyadenylation signal.

Another preferred embodiment of the present invention is amulticistronic vector as defined above, the vector additionallycomprising a cistron coding for a G-protein, preferably G-alpha 15, theg-protein preferably being located between the last GPCR and theselection marker and being functionally connected to both of them via anIRES element as defined supra.

A further preferred embodiment are cell lines transformed with thevectors of the present invention. Respective eukaryotic cell lines areamphibian, worm, insect or mammalian cells such as CHO, Hela, Hek-293and the like, e.g., cultured cells, explants, and cells in vivo.

Preferably, the host cell is selected from the group consisting of:HEK293 (human embryo kidney), Hela (Human Negroid cervix epitheloidcarcinoma), HT29 (Human Caucasian colon adenocarcinoma grade II), A431(human squamous carcinoma), IMR 32 (human caucasian neuroblastoma), K562(Human Caucasian chronic myelogenous leukaemia), U937 (Human Caucasianhistiocytic lymphoma), MDA-MB-231 (Human Caucasian breastadenocarcinoma), SK-N-BE(2) (Human Caucasian neuroblastoma), SH-SY5Y(Human neuroblastoma), HL60 (human promyelocytic leukemia) or eukaryoticnon-human cell lines like CHO-K1 (Hamster Chinese ovary), COS-7 (MonkeyAfrican green kidney, SV40 transformed), S49 (mouse lymphoma), Ltk(Mouse C34/An connective tissue), NG108-15 (Mouse neuroblastoma×Ratglioma hybrid), B50 (Rat nervous tissue neuronal, ECACC), C6 (Rat glialtumour), Jurkat (Human leukaemic T cell lymphoblast), BHK (HamsterSyrian kidney), Neuro-2a (Mouse Albino neuroblastoma), NIH/3T3 (mouseembryo fibroblast), preferably HEK293 (human embryo kidney), Hela (HumanNegroid cervix epitheloid carcinoma), CHO-K1 (Hamster Chinese ovary) orNeuro-2a (Mouse Albino neuroblastoma).

Further, preferred embodiments of the present invention are themodulators of GPCRs identifiable by the methods of the present inventionand further characterized by being selected from the group consisting ofsugars, steroids, tannins and lignans, terpenes, quinons, macrocycles,heterocycles, N-heterocycles and O-heterocycles, aliphatics andpolyketides, flavonoids, proteins, peptides and amino acids, alkaloidsand arenes, as stated above.

The intense investigations of the present inventors that led to theestablishment of the method of the present invention showed that therecombinant co-expression of said taste receptors as stable integrantsof eucaryotic host cells is hampered by a common instability. Althoughthe phenomenon is far away from being understood, it might be possiblethat the expression and specific signal transduction of these T1R-typereceptors interferes in a negative fashion with the cell physiology ofsuch stable host cells.

Within the context of the present invention, an instable integration ischaracterized by a rapid loss of functionality within the first 10passages of a selected recombinant cell line clone. Within these firstten passages it becomes obvious that more and more cells of a selectedrecombinant clone loose their characteristic functional taste GPCRexpression, indicated by passage dependent incremental decrease ofsweetener induced calcium inductions (Fluo-4 measurements). By way ofcontrast, the stable integrants of the present invention arecharacterized in that they show a functional expression of said tastereceptors at least over 50 passages, indicated by stable sweetenerinduced calcium inductions (Fluo-4 measurements) with a selected tastereceptors expressing cell line clone.

As becomes clear from the present specification of the invention, theuse and the hetero-oligomeric expression of the taste receptorcombinations T1R1/T1R3 and more preferred T1R2/T1R3 to identifycompounds as modulators of sweet taste in the field of tastants areespecially preferred embodiments of the present invention.

Accordingly, this invention relates to recombinantly engineered T1R-typeGPCRs having activity in cell based tastant assays, obtainable by (a)improving the coding sequence of the human sweet taste receptors (T1Rs)and/or (b) cloning them in a multicistronic expression unit forcoordinated expression and simultaneous selection for the generation ofstable eucaryotic cell systems; and/or (c) tagging one or both GPCRs inthe hetero-dimeric expression complex with G-proteins or G-Proteinchimeras to enable an improved fluorescence read out for theidentification of GPCR modulators, preferably sweet taste modulatingcompounds.

The receptor sequences applied for the here presented invention arefurthermore examplified in Example 1. By adding a prefix the optimizedreceptors have been termed shT1R2 and shT1R3. The respective amino acidsequences are depicted in SEQ ID NOs 1 and 2, the respective nucleicacid sequences in SEQ ID NOs 3 and 4.

A further means of the present invention to overcome the instability ofco-expressed GPCRs, preferably taste receptors T1R2 and T1R3 in stablecell line development is the construction of multicistronictranscription units. The method for obtaining a multicistronicexpression vector for the simultaneous expression of taste receptors anda selection marker of the invention is further examplified in Example 2.As stated above the development of T1R2/T1R3 expressing stable celllines e.g. Hek293, CHO, Hela for their use in cell based assay systemsis hampered by upcoming instabilities within the ongoing eucaryotic cellcultivation process and passaging numbers. For the generation of stablecell lines in the art most often expression vectors are used were theselection marker, conferring resistance to e.g. neomycin, hygromycin,zeocin, blasticidin or puromycin, is in fact coded on the same plasmidvector under the control of a independent promoter.

This situation can be avoided if the receptors and the selection markerare encoded in a multicistronic transcription unit.

However, so far multicistronic expression vectors have been describedonly for the use of inducible protein expression e.g. for gene therapyapplications (Fussenegger et al., 1998; Moser et al., 2000).

For the present invention the two taste receptors shT1R2 and shT1R3 havebeen cloned in the first and second position of a tricistronicexpression unit, whereas the deaminase gene conferring resistance to theselection marker blasticidin was cloned into the third position of theexpression unit. The tricistronic unit is under the control of thestrong human elongation factor 1 alpha promoter and the genes in thesecond and third position are preceded by an IRES element to facilitatetheir translation initiation in the resulting mRNA.

So far an eight kb multicistronic expression system for the stablefunctional expression of membrane located GPCRs has not been described.Usually these multicistronic systems—already in expressions of muchsmaller gene products like fluorescent proteins—are characterized by anon-stoichiometric and decreasing expression with increased distancefrom the promoter. Since the taste GPCRs significantly exceed the sizeof GFP or genes so far used in tri- or quadrocistronic expressionsystems; and the fact that the GPCRs have to be co-expressed to form aheterodimeric protein complex to function as sweetener responsivereceptors it was an unforeseeable and surprising result of the intenseinvestigations leading to the present invention that only the use ofmulticistronic vectors, i.e. tri- or tetracistronic vectors let tosatisfying results in establishing the screening method of the presentinvention.

In a further embodiment, this invention relates to methods using thebefore mentioned multicistronic expression vector to create stable celllines suited to express T1R polypeptides to construct a cell basedscreening tool for the search of novel compounds in the field of sweettaste modulators. Preferably, the cells comprise a functional G protein,e.g., G-alpha-15, G-alpha-16 or chimeric G protein like the ones withaltered c-terminus previously identified:

a) substitution of the last five amino acids of G-alpha 15:

-   -   G-alpha_(—)15 (EINLL), replaced with EYNLV (G-alpha q and        G-alpha 11), EFNLV (G-alpha 14), QYELL (G-alpha s and G-alpha        olf), DCGLF (G-alpha i1, G-alpha i2, G-alpha t1, G-alpha t2, and        G-alpha gust), ECGLY (G-alpha i3), GCGLY (G-alpha o1 and G-alpha        o2), YIGLC (G-alpha z), DIMLQ (G-alpha 12), QLMLQ (G-alpha 13)        or G-alpha 16 replaced with ECGLY (G-alpha 16 i3)        b) substitution of the last 44 carboxy-terminal amino acids of        G-alpha-16:    -   G-alpha 16gust44 (44 amino acids of the G-protein gustducin)    -   G-alpha 16z44 (44 amino acids of G-alpha-z);    -   G-alpha 16C44i2 (44 amino acids of the G-alpha-12);    -   G-alpha 16C44i3 (44 amino acids of the G-alpha-13);        or another G protein that is capable of coupling the chimeric        receptor to an intracellular signaling pathway or to a signaling        protein such as phospholipase C. (Li et al., 2002; Offermanns,        2003; Offermanns and Simon, 1995; Ueda et al., 2003). For this        purpose HEK 293 cells comprising a functional G protein have        been transfected with the vector pTrix-Eb_R2R3, cultured in the        presence of blasticidine, and stable cell lines have been        selected. As shown in Example 3 such cells have been found to        exhibit responses to taste stimuli and in addition these cell        lines have—compared to those derived with monocistronic T1R        expression vectors—a pronounced stable expression of the T1R        polypeptides. A stable cell line based on the expression vector        pTrix-Eb_R2R3 (see example 3) shows calcium responses to several        artificial sweeteners and a natural sweet protein. These        responses can still be measured after more than 50 passages of        the clone under selection with blasticidin and G418. Such        results could not be achieved with monocistronic or bicistronic        expressions. RT-PCR results of monocistronic clones revealed the        loss of both receptors with the initial 5 to 10 passages under        selection conditions. The stable cell line HEK_Ga15#17R2R3b #8        also shows dosis-dependent calcium responses for the above shown        sweet tastants which correlated with physiological taste        responses.

A further unexpected effect is that these multicistronic stable tastereceptor expressing cell lines are well suited for natural compoundscreening using complex microbial extract preparations. For theisolation and preparation of such complex screening samples standardmicrobial methods can be applied which encompass the cultivation ofmicroorganisms. Bioactive metabolites of interest from such cultivationsmay include intracellular molecules as well as those secreted into thecultivation media. Depending on the number of samples to be processed, achoice can generally be made from different options: (i) heating of thewhole broth; (ii) filtration of broth supernatant through a highmolecular weight exclusion filter with subsequent freeze-drying of thefiltrate; (iii) extraction of either the whole broth (ultrasonication,french press, cell disruption bombs or similar devices) or the brothsupernatant with organic solvents of varying polarity (e. g. chloroform,aceton, methanol or ethyl acetat) followed by evaporation of theextracts to dryness. (iv) Mixing the whole broth or the brothsupernatant with resins (e.g. AMBERLITE®, XAD-2, XAD-4, XAD-9 orsimilar) with subsequent solid phase extraction with organic solvents(e.g. acetone, acetic acid, butanol, ethanol, ethyl acetate, methanol,polyglycols or similar) either fractionated by applying increasingsolvent concentrations or as whole batch by applying the pure solventwithout fractionation. Solid phase extracts can be used directly insuited assay systems or further evaporated to dryness and resolved understandardized conditions for subsequent testing, e.g. the activation oftaste receptors in cell based assays. Activation of T1R receptors insuch cells can be detected using any standard methods, such as bydetecting changes in intracellular calcium by detecting Fluo-4 dependentfluorescence in the cell. Such an assay is the basis of the experimentalfindings presented in this application.

In a similar manner plant extracts well known to the skilled man may beused as well.

In FIG. 3 the activity of a stable cell line derived with themulticistronic expression plasmid pTrix-Eb-R2R3 in a G-alpha 15G-Protein background is depicted. The data of FIG. 3 document that thesecell line amongst several other isolated cell lines, shows T1R2/T1R3dependent activity in cell based assays towards several tastants likecyclamate, D-phenylalanine, saccharin, acesulfam K, aspartam andthaumatin.

In a further embodiment, this invention relates to the use ofT1R-G-protein fusion genes for the development of T1R-dependent cellbased assays. It has been shown that GPCRs can be fused in frame withdifferent polypeptides without loosing their functional activity(Milligan et al., 2003). From the present data it cannot be foreseenthat a fusion of a functional G-Protein to any GPCR leads to an improvedactivity compared to the wild type unfused situation. However it isscheduled to develop T1R2- and/or T1R3-fusion proteins in which theT1R-GPCR is fused to a functional G-Protein which is able to connect theT1R dependent signaling to the calcium inducing pathways used for T1Rdependent activity measurement in cell based assays as exemplified inExample 3. Preferably, the T1R-fusion proteins comprise a functional Gprotein, e.g., Galpha-15 or chimeric G protein like the ones previouslyidentified, or another G protein that is capable of coupling thechimeric receptor to an intracellular signaling pathway or to asignaling protein such as phospholipase C. (Offermanns, 2003; Offermannsand Simon, 1995; Ueda et al., 2003) For the development of such cellbased assays the fusion proteins will be subcloned into themulticistronic expression vector backbone leading to expression vectorof the type pTrix-Eb-R2Gq-R3 depicted in FIG. 4.

The figures show:

FIG. 1: Bioinformatic alignment with Clustal X software: Different GPCRnucleotide sequences of the T1R class are presented in an alignmentanalysis. Human wild type cDNA sequences are depicted as wt_hT1Rs; thetheoretically most optimized cDNA sequences after multi parameteroptimization are depicted as opt_hT1Rs; partly optimized cDNAs used forstable cell line development are termed sh_T1Rs. The nucleotidealignments are presented as phylogenetic trees in which wt_T1R1 wasdefined as the outgroup. The underlying distance matrix is shown below.

FIG. 2: Multicistronic eucaryotic expression vector pTrix-Eb-R2R3: Theexpression of the taste receptor genes shT1R2, shT1R3 and blasticidin Sdeaminase (bsd) gene are under the control of the human elongationfactor 1 alpha promoter (PEF1a). To confer multicistronic expression onthe translational level two internal ribosomal entry sites (Cite I andCite II) have been inserted. The multicistronic unit is terminated by asimian virus 40 polyadenylation site. The prokaryotic origin ofreplication (ori) and the kanamycin resistance gene serve for thepropagation, amplification and selection of the plasmid vector in E.coli.

FIG. 3: The stable HEK_Ga15#17R2R3b #8 was selected with blasticidin andG418 after transfection of the stable HEK_Ga15#17 (G418) withpTriX_Eb_R2R3. This tricistronic expression was necessary for theselection and retrieval of stable clones. Clone #8 shows calciumresponses to several artificial sweeteners and a natural sweet protein.

FIG. 4: Multicistronic eucaryotic expression vector pTrix-Eb-R2R3: Theexpression of the taste receptor genes shT1R2-Gq, shT1R3 and blasticidinS deaminase (bsd) gene are under the control of the human elongationfactor 1 alpha promoter (PEF1a). To confer multicistronic expression onthe translational level two internal ribosomal entry sites (Cite I andCite II) have been inserted. The multicistronic unit is terminated by asimian virus 40 polyadenylation site. The prokaryotic origin ofreplication (ori) and the kanamycin resistance gene serve for thepropagation, amplification and selection of the plasmid vector in E.coli.

EXPERIMENTAL MATERIALS AND METHODS Cell Culture

Transient transfection/selection of stable HEK293 cells—Transient andstable transfections can be performed with lipid complexes like calciumphosphate precipitation, Lipofectamine/PLUS reagent (Invitrogen),Lipofectamine 2000 (Invitrogen) or MIRUS TransIT293 (Mirus BioCorporation) according to the manuals. Electroporation can also be amethod of choice for stable transfection of eukaryotic cells.

The cells are seeded in 6-well plates at a density of 4×105 cells/well.HEK293 cells are transfected with linearised plasmids for stableexpression of the genes of interest. After 24 hours, the selection withselecting reagents like zeocin, hygromycin, neomycin or blasticidinstarts. About 50 μl to 300 μl trypsinized transfected cells from a6-well are seeded in a 100 mm dish and the necessary antibiotic is addedin an appropriate concentration. Cells are cultivated until clones arevisible on the 100 mm cell culture plate. These clones are selected forfurther cultivation and calcium imaging. It takes about four to eightweeks to select cell clones which stably express the genes of interest.

Calcium Imaging

Fluo-4 AM assay with stable HEK293 cells—Stable cells are maintained inDMEM high-glucose medium (Invitrogen) supplemented with 10% fetal bovineserum (Biochrom) and 4 mM L-glutamine (Invitrogen). Cells for calciumimaging are maintained in DMEM low-glucose medium supplemented with 10%FBS and 1× Glutamax-1 (Invitrogen) for 48 hours before seeding. Thesestable cells are trypsinized after 48 hours (either with Trypsin-EDTA,Accutase or TrypLE) and seeded onto poly-D-lysine coated 96-well assayplates (Corning) at a density of 45,000 cells/well in DMEM low-glucosemedium supplemented with 10% FBS and 1× Glutamax-1.

After 24 hours, the cells were loaded in 100 μl medium with additional100 μl of 4 μM Fluo-4 (calcium sensing dye, 2 μM end concentration;Molecular Probes) in Krebs-HEPES (KH)-buffer for 1 hour. The loadingreagent is then replaced by 80 μl KH-buffer per well. TheKrebs-HEPES-buffer is a physiological saline solution including 1.2 mMCaCl2, 4.2 mM NaHCO3 and 10 mM HEPES.

The dye-loaded stable cells in plates were placed into a fluorescencemicrotiter plate reader to monitor fluorescence (excitation 488 nm,emission 520 nm) change after the addition of 20 μl KH-buffersupplemented with 5× tastants. For each trace, tastant was added 11.5seconds after the start of the scan and mixed two times with the buffer,scanning continued for an additional 32 seconds, and data were collectedevery second.

Data analysis/Data recording—Calcium mobilization was quantified as thechange of peak fluorescence (ΔF) over the baseline level (F). Data wereexpressed as the mean S.E. of the ΔF/F value of replicated independentsamples. The analysis was done with the software of the microtiter platereader.

EXAMPLES

The following examples are provided to illustrate preferred embodimentsand are intended to be illustrative and not limitative of the scope ofthe invention. In the DNA sequences presented herein, the one lettercodes N or n refers to any of the of the four common nucleotide bases,A, T, C, or G. In the protein sequences presented herein, the one-lettercode X or Xaa refers to any of the twenty common amino acid residues.

Example 1 Synthesis and Design of Synthetic Intronless hT1R2 and hT1R3cDNAs

The nucleotide sequence of the human receptors hT1R2 and hT1R3 are basedon their wild type coding DNAs and have been optimized according to amultiparameter analysis considering optimal codon usage, putativecryptic splice sites, putative repeated sequences as well as AT-rich orGC-rich sequence stretches. Gene optimization often has favorableeffects on enhanced mRNA stability, translational efficiency and reducedRNA secondary structure to prevent transcriptional pausing or prematuretermination of transcriptional elongation.

Up to now the binding sites for only a few ligands of the sweet tasteheterodimeric receptor T1R2/T1R3 are known. Hence, the encoded wild-typeamino acid sequences of the receptors were left unchanged in thismultiparameter optimization, to retain the receptors binding qualities.

Synthesis and construction of the receptor cDNAs was done via theassembly of synthetic oligonucleotides and subsequent cloning into astandard pUC-18 plasmid vector for further amplification. Alternatively,these nucleic acids can be cloned in vitro by well-known cloningtechniques, (Ausubel et al., 1998; Pachuk et al., 2000; Sambrook et al.,1989; Stemmer et al., 1995). Double-stranded DNA fragments may then beobtained either by synthesizing the complementary strand and annealingthe strands together under appropriate conditions, or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

Due to the synthetic generation of the cDNAs the receptors of thepresent invention have been described with the prefix “s” and termedshT1R2 and shT1R3. The nucleic acid and amino acid sequences for theabove mentioned T1R cloned sequences as well as other full-length andpartial T1R sequences are set forth in the sequence protocoll:

Example 2 Multicistronic Vectors for the Expression of Taste Receptors

For the construction of a multicistronic expression unit the tastereceptor sequences displayed in Example 1 have been used. As shown inFIG. 2 the tricistronic expression unit is under the control of thehuman elongation factor 1 alpha promoter. Using standard cloningtechniques the cDNA for the receptors shT1R2 and shT1R3 and the cDNA forthe blasticidin S deaminase gene have been cloned. To enable thetranslation initiation of each gene of this tricistronic unit twoEMC-virus derived internal ribosomal entry sites (IRES—also termedCap-independent translation enhancer (CITE)) have been inserted.(Jackson et al., 1990; Jang et al., 1988) The tricistronic expressionunit is terminated by a simian virus 40 polyadenylation signal sequence.This composition permits the simultaneous expression of all three genesunder the control of only one promoter. In contrast to monocistronictranscription units, which integrate independently from each other intodifferent chromosomal locations during the process of stable cell linedevelopment, the tricistronic transcription unit integrates allcontaining genes in one and the same chromosomal locus. Due to thealignment of the genes, the blasticidin S deaminase gene is onlytranscribed in case a full length transcription takes place. Moreoverthe polarity of multicistronic transcription units (Moser et al., 2000)leads probably to a balanced stoichiometry of the receptor genes andtheir expression rates in the range of 1:0.7 up to 1:1 for the first twopositions whereas the blasticidin S deaminase gene compared to thereceptor genes in the third position is expressed to a lesser extend.Assuming that for the functional heterodimeric receptor shT1R2/shT1R3 a1:1 stoichiometry is needed the lesser polarity effects for the receptorgenes promote the desired stoichiometry whereas the reduced expressionof the deaminase promotes an integration locus with enhancedtranscriptional activity.

Example 3 Detection of T1R2/T1R3 Dependent Activity

In wild type taste cells—e.g. in the human taste bud—signal transductionis presumably transduced by the G-proteins gustducin and/or byG-Proteins of the Galpha-i type. Encountering sweet ligands theheterodimeric taste receptor T1R2/T1R3 reacts with induction of secondmessenger molecules; either induction of the cAMP level in response tomost sugars or induction of the calcium level in response to mostartificial sweeteners. (Margolskee, 2002)

To analyze the function and activity of the heterodimeric T1R2/T1R3taste receptor a calcium dependent cell based assay has been utilized.Briefly, synthetic T1R type taste receptors (as shown in Example 1) havebeen transfected with the plasmid vector pTrix-Eb-R2R3 (see Example 2)in a HEK293 cell line stably expressing the mouse G-alpha-15 G-protein.Selection of T1R2/T1R3 expressing cells has been performed by culturingthe transfected cells in the presence of blasticidine.

For measurement of T1R2/T1R3 taste receptor dependent activity HEK293cells stably expressing G-alpha-15, shT1R2 and shT1R3 were 4×104 seededin 96-well plates and labeled with the calcium sensitive fluorescencedye Fluo4-AM (2 μM) in DMEM culture medium for one hour at 37° C. Forthe measurement in a fluorescence plate reader the medium was exchangedfor KH-buffer and incubated for another 15 minutes at 37° C.Fluorescence measurement of the labeled cells was conducted in aNovostar fluorescence plate reader (BMG, Offenburg, Germany). Responseto different tastants as depicted in FIG. 3 was recorded as Fluo4-AMfluorescence increase initiated through the T1R2/T1R3 dependent increaseof the second messenger calcium. After obtaining calcium signals foreach sample, calcium mobilization in response to tastants was quantifiedas the relative change (peak fluorescence F1−baseline fluorescence F0level, denoted as dF) from its own baseline fluorescence level (denotedas F0). Though rel. RFU is dF/F0. Peak fluorescence intensity occurredabout 20-30 sec after addition of tastants. The data shown were obtainedfrom two independent experiments and done in triplicates.

REFERENCES

-   Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,    Seidman, J. G., Smith, J. A., and Struhl, K. (1998). /Current    protocols in molecular biolong. //V. B. Chanada, series ed. //New    York: Wiley & Sons./-   Bachmanov, A. A., Tordoff, M. G., and Beauchamp, G. K. (2001).    Sweetener preference of C57BL/6ByJ and 129P3 /J mice. /Chem Senses/    26, 905-13.-   Bai, M., Trivedi, S., and Brown, E. M. (1998). Dimerization of the    extracellular calcium-sensing receptor (CaR) on the cell surface of    CaR-transfected HEK293 cells. /J Biol Chem/ 273, 23605-10.-   Bockaert, J., and Pin, J. P. (1999). Molecular tinkering of G    protein-coupled receptors: an evolutionary success. /Embo J/ 18,    1723-9.-   Buck, L., and Axel, R. (1991). A novel multigene family may encode    odorant receptors: a molecular basis for odor recognition. /Cell/    65, 175-87.-   Eglen, R. (2005). An Overview of High Throughput Screening at G    Protein Coupled Receptors. /Frontiers in Drug Design & Discovery/ 1,    97-111.-   Filmore, D. (2004). It's a GPCR world. /Modern Drug Discovery/,    24-28.-   Firestein, S. (2001). How the olfactory system makes sense of    scents. /Nature/ 413, 211-8.-   Fussenegger, M., Mazur, X., and Bailey, J. E. (1998). pTRIDENT, a    novel vector family for tricistronic gene expression in mammalian    cells. /Biotechnol Bioeng/ 57, 1-10.-   Fux, C., Langer, D., Kelm, J. M., Weber, W., and Fussenegger, M.    (2004). New-generation multicistronic expression platform: pTRIDENT    vectors containing size-optimized IRES elements enable homing    endonuclease-based cistron swapping into lentiviral expression    vectors. /Biotechnol Bioeng/ 86, 174-87.-   George, S. R., O'Dowd, B. F., and Lee, S. P. (2002).    G-protein-coupled receptor oligomerization and its potential for    drug discovery. /Nat Rev Drug Discov/ 1, 808-20.-   Grenby, T. H. (1996): Advances in Sweeteners, 1^(st) edition,    Blackie Academic & Professional, London.-   Hartenbach, S., and Fussenegger, M. (2005). Autoregulated,    bidirectional and multicistronic gas-inducible mammalian as well as    lentiviral expression vectors. /J Biotechnol/.-   Hellen, C. U., and Sarnow, P. (2001). Internal ribosome entry sites    in eukaryotic mRNA molecules. /Genes Dev/ 15, 1593-612.-   Hoon, M. A., Adler, E., Lindemeier, J., Battey, J. F., Ryba, N. J.,    and Zuker, C. S. (1999). Putative mammalian taste receptors: a class    of taste-specific GPCRs with distinct topographic selectivity.    /Cell/ 96, 541-51.-   Hoon, M. A., and Ryba, N. J. (1997). Analysis and comparison of    partial sequences of clones from a taste-bud-enriched cDNA library.    /J Dent Res/ 76, 831-8.-   Jackson, R. J., Howell, M. T., and Kaminski, A. (1990). The novel    mechanism of initiation of picornavirus RNA translation. /Trends    Biochem Sci/ 15, 477-83.-   Jang, S. K., Krausslich, H. G., Nicklin, M. J., Duke, G. M.,    Palmenberg, A. C., and Wimmer, E. (1988). A segment of the 5′    nontranslated region of encephalomyocarditis virus RNA directs    internal entry of ribosomes during in vitro translation. /J Virol/    62, 2636-43.-   Kaupmann, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W.,    Beck, P., Mosbacher, J., Bischoff, S., Kulik, A., Shigemoto, R.,    Karschin, A., and Bettler, B. (1998). GABA(B)-receptor subtypes    assemble into functional heteromeric complexes. /Nature/ 396, 683-7.-   Kinnamon, S. C., and Cummings, T. A. (1992). Chemosensory    transduction mechanisms in taste. /Annu Rev Physiol/ 54, 715-31.-   Kinnamon, S. C., and Margolskee, R. F. (1996). Mechanisms of taste    transduction. /Curr Opin Neurobiol/ 6, 506-13.-   Kitagawa, M., Kusakabe, Y., Miura, H., Ninomiya, Y., and Hino, A.    (2001). Molecular genetic identification of a candidate receptor    gene for sweet taste. /Biochem Biophys Res Commun/ 283, 236-42.-   Kramer, B. P., Weber, W., and Fussenegger, M. (2003). Artificial    regulatory networks and cascades for discrete multilevel transgene    control in mammalian cells. /Biotechnol Bioeng/ 83, 810-20.-   Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M.,    Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000).    Structural basis of glutamate recognition by a dimeric metabotropic    glutamate receptor. /Nature/ 407, 971-7.-   Leatherhead Food R A (2000). Ingredients Handbook Sweeteners, 2^(nd)    edition, Leatherhead Publishing, Leatherhead, Surrey.-   Li, X., Inoue, M., Reed, D. R., Huque, T., Puchalski, R. B.,    Tordoff, M. G., Ninomiya, Y., Beauchamp, G. K., and Bachmanov, A. A.    (2001). High-resolution genetic mapping of the saccharin preference    locus (Sac) and the putative sweet taste receptor (T1R1) gene    (Gpr70) to mouse distal Chromosome 4. /Mamm Genome/ 12, 13-6.-   Li, X., Staszewski, L., Xu, H., Durick, K., Zoller, M., and    Adler, E. (2002). Human receptors for sweet and umami taste. /Proc    Natl Acad Sci USA/ 99, 4692-6.-   Lindemann, B. (1996a). Chemoreception: tasting the sweet and the    bitter. /Curr Biol/ 6, 1234-7.-   Lindemann, B. (1996b). Taste reception. /Physiol Rev/ 76, 718-66.-   Lindemann, B. (2001). Receptors and transduction in taste. /Nature/    413, 219-25.-   Lush, I. E. (1989). The genetics of tasting in mice. VI. Saccharin,    acesulfame, dulcin and sucrose. /Genet Res/ 53, 95-9.-   Lush, I. E., Hornigold, N., King, P., and Stoye, J. P. (1995). The    genetics of tasting in mice. VII. Glycine revisited, and the    chromosomal location of Sac and Soa. /Genet Res/ 66, 167-74.-   Maeda, T., Imanishi, Y., and Palczewski, K. (2003). Rhodopsin    phosphorylation: 30 years later. /Prog Retin Eye Res/ 22, 417-34.-   Margolskee, R. F. (2002). Molecular mechanisms of bitter and sweet    taste transduction. /J Biol Chem/ 277, 1-4.-   Max, M., Shanker, Y. G., Huang, L., Rong, M., Liu, Z., Campagne, F.,    Weinstein, H., Damak, S., and Margolskee, R. F. (2001). Tas1r3,    encoding a new candidate taste receptor, is allelic to the sweet    responsiveness locus Sac. /Nat Genet/ 28, 58-63.-   McLaughlin, S. K., McKinnon, P. J., and Margolskee, R. F. (1992).    Gustducin is a taste-cell-specific G protein closely related to the    transducins. /Nature/ 357, 563-9.-   Meyerhof, W., Behrens, M., Brockhoff, A., Bufe, B., and Kuhn, C.    (2005). Human bitter taste perception. /Chem Senses/ 30 Suppl 1,    i14-i15. Milligan, G. (2003). High-content assays for ligand    regulation of G-protein-coupled receptors. /Drug Discov Today/ 8,    579-85.-   Milligan, G., Ramsay, D., Pascal, G., and Carrillo, J. J. (2003).    GPCR dimerisation. /Life Sci/ 74, 181-8.-   Montmayeur, J. P., Liberles, S. D., Matsunami, H., and Buck, L. B.    (2001). A candidate taste receptor gene near a sweet taste locus.    /Nat Neurosci/ 4, 492-8.-   Moser, S., Schlatter, S., Fux, C., Rimann, M., Bailey, J. E., and    Fussenegger, M. (2000). An update of pTRIDENT multicistronic    expression vectors: pTRIDENTs containing novel    streptogramin-responsive promoters. /Biotechnol Prog/ 16, 724-35.-   Nelson, G., Chandrashekar, J., Hoon, M. A., Feng, L., Zhao, G.,    Ryba, N. J., and Zuker, C. S. (2002). An amino-acid taste receptor.    /Nature/ 416, 199-202.-   Nelson, G., Hoon, M. A., Chandrashekar, J., Zhang, Y., Ryba, N. J.,    and Zuker, C. S. (2001). Mammalian sweet taste receptors. /Cell/    106, 381-90.-   O'Brien Nabors, L. (2001). Alternative Sweeteners, 3^(rd) edition,    Marcel Dekker, Inc., New York, Basel.-   Offermanns, S. (2003). G-proteins as transducers in transmembrane    signalling. /Prog Biophys Mol Biol/ 83, 101-30.-   Offermanns, S., and Simon, M. I. (1995). G alpha 15 and G alpha 16    couple a wide variety of receptors to phospholipase C. /J Biol Chem/    270, 15175-80.-   Pachuk, C. J., Samuel, M., Zurawski, J. A., Snyder, L., Phillips,    P., and Satishchandran, C. (2000). Chain reaction cloning: a    one-step method for directional ligation of multiple DNA fragments.    /Gene/ 243, 19-25.-   Pierce, K. L., Premont, R. T., and Lefkowitz, R. J. (2002).    Seven-transmembrane receptors. /Nat Rev Mol Cell Biol/ 3, 639-50.-   Pin, J. P., Galvez, T., and Prezeau, L. (2003). Evolution,    structure, and activation mechanism of family 3/C G-protein-coupled    receptors. /Pharmacol Ther/ 98, 325-54.-   Sainz, E., Korley, J. N., Battey, J. F., and Sullivan, S. L. (2001).    Identification of a novel member of the T1R family of putative taste    receptors. /J Neurochem/ 77, 896-903.-   Salahpour, A., Angers, S., and Bouvier, M. (2000). Functional    significance of oligomerization of G-protein-coupled receptors.    /Trends Endocrinol Metab/ 11, 163-8.-   Sambrook, J., Fritsch, E., and Maniatis, T. (1989). Molecular    cloning. A Laboratory Manual. /In: second ed. //Cold Spring Harbor    Laboratory Press. Cold Spring Harbor. NY./-   Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M., and    Heyneker, H. L. (1995). Single-step assembly of a gene and entire    plasmid from large numbers of oligodeoxyribonucleotides. /Gene/ 164,    49-53.-   Sugita, M., and Shiba, Y. (2005). Genetic tracing shows segregation    of taste neuronal circuitries for bitter and sweet. /Science/ 309,    781-5.-   Ueda, T., Ugawa, S., Yamamura, H., Imaizumi, Y., and Shimada, S.    (2003). Functional interaction between T2R taste receptors and    G-protein alpha subunits expressed in taste receptor cells. /J    Neurosci/ 23, 7376-80.-   Von Rymon Lipinski, G.-W., Schiweck, H. (1991). Handbuch    Süβungsmittel—Eigenschaften und Anwendung, Behr's Verlag, Hamburg.-   Weber, W., Malphettes, L., de Jesus, M., Schoenmakers, R.,    El-Baba, M. D., Spielmann, M., Keller, B., Weber, C. C., van de    Wetering, P., Aubel, D., Wurm, F. M., and Fussenegger, M. (2005).    Engineered Streptomyces quorum-sensing components enable inducible    siRNA-mediated translation control in mammalian cells and adjustable    transcription control in mice. /J Gene Med/ 7, 518-25.-   White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J.,    Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M., and    Marshall, F. H. (1998). Heterodimerization is required for the    formation of a functional GABA(B) receptor. /Nature/ 396, 679-82.-   Wong, G. T., Gannon, K. S., and Margolskee, R. F. (1996).    Transduction of bitter and sweet taste by gustducin. /Nature/ 381,    796-800.-   Zhao, F. L., Lu, S. G., and Herness, S. (2002). Dual actions of    caffeine on voltage-dependent currents and intracellular calcium in    taste receptor cells. /Am J Physiol Regul Integr Comp Physiol/ 283,    R115-29.

1. Multiparameter optimized nucleic acid molecule coding for a GPCR,wherein the nucleic acid molecule is SEQ ID NO
 4. 2. Nucleic acidmolecules functionally equivalent to the molecules according to claim 1.3. Multicistronic expression vector comprising more than one cistroncoding for a GPCR.
 4. Cell lines stably transfected with a nucleic acidmolecule according to claim
 1. 5. Multiparameter optimized nucleic acidmolecule according to claim 1, wherein the GPCR is a T1R-type GPCR. 6.Cell lines stably transfected with a vector according to claim 3.